Gliomas are the most common primary tumors of the central nervous system (CNS) {Louis, 1995}. Glioblastoma multiforme (GBM) is the most frequent and the most malignant type of glioma. There is a much higher incidence of GBM in adults than in children. According to the Central Brain Tumor Registry of the United States statistical report, GBM accounts for about 20% of all brain tumors in the USA (CBTRUS, 19982002). Current treatment for patients with GBM include, surgery followed by radiation and chemotherapy. Despite intensive research the median survival for GBM patients until the early 1990s was less than a year {Walker, 1978}. The single most important advance in the treatment of these tumors over the past 30 years has been the introduction of temozolomide, initially in combination with external beam irradiation, and then followed by repetitive cycles of temozolomide alone {Stupp, 2007}. However, this has increased the overall median survival by only 75 days. Clearly, new and more efficient therapeutic approaches are needed to improve GBM patient survival. Monoclonal antibodies (mAbs), either armed (fused to immunotoxin [IT] or radioisotopes) or unarmed, are presently a rapidly growing category of new drug entities. This is well demonstrated by the large number of mAb-based clinical trials currently in progress for brain tumor patients. Boskovitz, A., Wikstrand, C. J., Kuan, C. T., Zalutsky, M. R., Reardon, D. A., and Bigner, D. D. Monoclonal antibodies for brain tumour treatment. Expert Opin Biol Ther, 4: 1453-1471, 2004. More recently, genetically engineered single-chain variable-region antibody fragments (scFvs), consisting of the heavy- and light-chain variable regions (VH and VL) fused to toxins and targeting antigens expressed specifically by brain tumor, are under investigation. Archer, G. E., Sampson, J. H., Lorimer, I. A., McLendon, R. E., Kuan, C. T., Friedman, A. H., Friedman, H. S., Pastan, I. H., and Bigner, D. D. Regional treatment of epidermal growth factor receptor vIII-expressing neoplastic meningitis with a single-chain immunotoxin, MR-1. Clin Cancer Res, 5: 2646-2652, 1999. Because it is small, an scFv-IT fusion protein should have greater tumor penetration than an intact IgG and therefore lead to enhanced therapeutic efficacy {Pastan, 1995}.
The epidermal growth factor receptor (EGFR) is a 170-kDa, transmembrane receptor tyrosine kinase (RTK). It is stimulated by binding of its ligands, such as transforming growth factor (TGF)-a or EGF, to its extracellular domain. Ligand binding induces receptor dimerization and activates a tyrosine-specific protein kinase activity {Ushiro, 1980} involved in controlling epithelial cell growth and proliferation. Ultimately, the receptor-ligand complexes are internalized, and the EGFR signal is terminated. EGFR overexpression is frequently observed in a wide variety of human cancers, including breast {Klijn, 1992; Osaki, 1992}, lung {Pavelic, 1993}, head and neck {Rubin Grandis, 1996}, prostate {Fox, 1994}, bladder {Chow, 2001}, colorectal {Yasui, 1988}, and ovarian carcinoma {Bartlett, 1996}, as well as brain tumors {Arita, 1989; Libermann, 1984}. In contrast, the level of EGFR in normal brain is undetectable or extremely low. EGFR is the most frequently amplified gene in GBM {Fuller, 1992}. Correlating with the gene amplification, the protein is overexpressed in about 60% to 90% of GBM cases. In the absence of gene amplification, protein overexpression has also been observed in 12% to 38% of GBM patients {Chaffanet, 1992}, which could be due to aberrant translational and post-translational mechanisms. Preclinical studies have shown that EGFR activation, in addition to protecting cells from apoptosis, also induces several tumorigenic processes, including proliferation, angiogenesis, and metastasis {Huang, 1999}.
EGFR gene amplification is often associated with gene rearrangements. Several EGFR deletion mutants have been identified {Rasheed, 1999}, the most common one being EGFRvIII, which is present in 20% to 50% of GBMs with EGFR amplification. Wikstrand, C. J., Fung, K. M., Trojanowski, J. Q., McLendon, R. E., and Bigner, D. D. Antibodies and molecular immunology: immunohistochemistry and antigens of diagnostic significance. In: D. D. Bigner, R. E. McLendon, and J. M. Bruner (eds.), Russell and Rubinstein's Pathology of the Nervous System, 6th edition, pp. 251-304. New York: Oxford University Press, 1998. The mutant EGFRvIII contains a deletion of exon 2-7 of the EGFR gene, which is characterized by an in-frame deletion of 801 base pairs of the coding region {Sugawa, 1990}. This deletion creates a novel glycine residue at the fusion junction at position 6, between amino acid residues 5 and 274, generating a tumor-specific protein sequence that is expressed specifically on tumor cells but not on normal tissues. EGFRvIII is a constitutively active RTK which is not further activated by EGFR ligands. Batra, S. K., Castelino-Prabhu, S., Wikstrand, C. J., Zhu, X., Humphrey, P. A., Friedman, H. S., and Bigner, D. D. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ, 6: 1251-1259, 1995. EGFRvIII is widely expressed in malignant gliomas {Humphrey, 1990} and carcinomas, including head and neck {Sok, 2006} and breast. Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey, P. A., Kurpad, S. N., McLendon, R. E., Moscatello, D., Pegram, C. N., Reist, C. J., and et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res, 55: 3140-3148, 1995. Overexpression of EGFRvIII induces resistance in glioma cells to commonly used chemotherapeutic agents {Nagane, 1998}.
Monoclonal antibodies targeting either the wild-type EGFR (EGFRwt) or EGFRvIII have been developed. One of them, D2C7, a murine IgG1κ, was developed by our group. The D2C7 hybridoma recognizes both the EGFRwt and the tumor-specific EGFRvIII receptors {Boskovitz, 2005}.
There is a continuing need in the art for effective means of treating brain tumors and prolonging life of affected patients.